Co-Delivery of Trichosanthin and Albendazole by Nano-Self-Assembly

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Co-Delivery of Trichosanthin and Albendazole by Nano-SelfAssembly for Overcoming Tumor Multidrug-Resistance and Metastasis Yisi Tang,†,‡,⊥ Jianming Liang,‡,§,⊥ Aihua Wu,†,‡ Yingzhi Chen,‡ Pengfei Zhao,‡ Tingting Lin,‡,∥ Meng Zhang,‡ Qin Xu,*,† Jianxin Wang,*,†,§ and Yongzhuo Huang*,‡ †

Guangzhou University of Chinese Medicine, 12 Ji-chang Road, Guangzhou 510450, China Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China § Department of Pharmaceutics, Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, School of Pharmacy, Fudan University, Shanghai 201203, China ∥ Department of Pharmacy, Binzhou Medical University Hospital, Binzhou 256603, China ‡

S Supporting Information *

ABSTRACT: Multidrug resistance (MDR) and metastasis are the major obstacles in cancer chemotherapy. Nanotechnology-based combination therapy is a useful strategy. Recently, the combination of biologics and small drugs has attracted much attention in cancer therapy. Yet, the treatment outcomes are often compromised by the different pharmacokinetic profiles of the co-administered drugs thus leading to inconsistent drug uptake and suboptimal drug combination at the tumor sites. Nanotechnology-based co-delivery offers a promising method to address this problem, which is well demonstrated in the use of small drug combinations. However, co-delivery of the drugs bearing different physicochemical properties (e.g., proteins and small drugs) remains a formidable challenge. Herein, we developed a self-assembled nanosystem for co-delivery of trichosanthin (TCS) protein and albendazole (ABZ) as a combination therapy for overcoming MDR and metastasis. TCS is a ribosome-inactivating protein with high antitumor activity. However, the druggability of TCS is poor due to its short half-life, lack of tumorspecific action, and low cell uptake. ABZ is a clinically used antihelmintic drug, which can also inhibit tubulin polymerization and thus serve as a potential antitumor drug. In our work, ABZ was encapsulated in the albumin-coated silver nanoparticles (termed ABZ@BSA/Ag NP). The thus-formed NPs were negatively charged and could tightly bind with the cationic TCS that was modified with a cell-penetrating peptide (CPP) low-molecular-weight protamine (termed rTL). Via the stable charge interaction, the nanosystem (rTL/ABZ@BSA/Ag NP) was self-assembled, and featured by the TCS corona. The co-delivery system efficiently inhibited the proliferation of the drug-resistant tumor cells (A549/T and HCT8/ADR) by impairing the cytoskeleton, arresting the cell cycle, and enhancing apoptosis. In addition, the migration and invasion of tumor cells were inhibited presumably due to the impeded cytoskeleton functions. The anti-MDR effect was further confirmed by the in vivo studies with the subcutaneous A549/T tumor mouse model. More importantly, the co-delivery system was demonstrated to be able to inhibit metastasis. The co-delivery system of TCS/ABZ provided a potential strategy for both overcoming drug resistance and inhibiting tumor metastasis. KEYWORDS: Multidrug resistance, trichosanthin, albendazole, albumin nanoparticles, silver nanoparticles, metastasis

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INTRODUCTION

essential method to overcome MDR and metastasis, yet the treatment outcomes are compromised by the different pharmacokinetic profiles of the co-applied drugs thus leading to inconsistent drug uptake and suboptimal drug combination at the tumor sites.5 This could be a major reason for the inconsistent correlation between cellular and animal results. Indeed, a drug combination screened by in vitro tests often

Multidrug resistance (MDR) and metastasis represent the formidable barricades in cancer pharmacotherapy. MDR involves various well-documented mechanisms, including overexpression of drug-efflux transporters, DNA-damage repair, and dysfunction of apoptosis.1 Recently, the connection between cytoskeleton alternations and drug resistance has attracted much attention.2 The cytoskeleton is also closely related to tumor-cell migration and metastasis.3 Therefore, the cytoskeleton could be a potential target to address both the MDR and metastasis issues.4 Combination therapy is an © 2017 American Chemical Society

Received: April 15, 2017 Accepted: July 25, 2017 Published: July 25, 2017 26648

DOI: 10.1021/acsami.7b05292 ACS Appl. Mater. Interfaces 2017, 9, 26648−26664

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of Tumor Delivery and Synergistic Effect via Co-Delivery of TCS and ABZ by Silver Nanoparticles

addition, rTL was incorporated into a self-assembled nanosystem so as to improve its half-life and tumor targeting. Albendazole (ABZ) is an anthelmintic drug, and its antiproliferative effect in helminths is associated with binding to β-tubulin to inhibit microtubule polymerization. As a βtubulin inhibitor, ABZ also exhibits potent antitumor activity.19 Therefore, we expected that TCS and ABZ could work synergistically on the inhibition of β-tubulin and yield an enhanced treatment effect. Here, we reported a novel combination nanotherapy for co-delivery of TCS and ABZ via albumin-based nanoparticles (NPs) (Scheme 1). The codelivery system was characterized by self-assembling via stable charge interaction between the cationic rTL and the anionic ABZ@BSA NPs. Two kinds of albumin-based NPs were prepared, that is, the albumin NPs and albumin-coated silver NPs, with encapsulation of ABZ, termed ABZ@BSA NPs and ABZ@BSA/Ag NPs, respectively. Targeting the upregulated signals in a tumor offers a promising method to improve the drug-delivery efficiency.20,21 In cancer cells, albumin is an essential source of energy and amino acids. The expression of albumin-binding proteins is upregulated in many types of solid tumors for promoting albumin uptake, and thereby they serve as a potential portal for cancer-drug transport.16 A benefit of the albumin-based nanocarriers is that they can potentiate endocytosis via the albumin-binding proteins-mediated pathway.

does not exhibit an expected in vivo efficacy. Co-delivery techniques based on nanotechnology could be ideal for combination therapy because of the ability to simultaneously carry the drugs to the same destination.6 In the recent decade, nanotechnology-based drug-delivery systems have been developed to overcome MDR via bypassing drug-efflux pumps.7 A nanodrug benefits from its size-exclusion effect, being too large to be pumped out by the MDR-related transporters.8 Taking P-glycoproteins (P-gp) for example, its transport substrates are limited to the compounds with a molecular weight no greater than 2000 Da.9 In this regard, protein drugs are a class of potent drugs against MDR due to their resistance to P-gp mediated efflux. It should be noted that the combined use of proteins and small drugs has become more and more common in clinical practice. However, co-delivery of the combination drugs bearing different physicochemical properties (e.g., proteins and small drugs) remains a huge challenge. Trichosanthin (TCS) is a type I ribosome-inactivating protein derived from the Chinese herb Tian Hua Fen, the root of Trichosanthes kirilowii Maxim. TCS is a prescription drug in China for gynecological use in ectopic pregnancies, hydatidiform moles, chorionic epithelioma, and abortions.10 TCS possesses high antitumor activity via the ribosomeinactivating mechanism and apoptosis induction thus displaying great promise for cancer therapy.11 Of interest, the anticancer activities of TCS are also associated with the induction of specific changes of the cytoskeleton configuration by reducing the expression of tubulins.12 We previously reported that TCS in combination with paclitaxel can efficiently reverse MDR, revealing its potential for anti-MDR cancer therapy.13 However, the antitumor application of TCS is restrained by its short halflife owing to rapid renal clearance related to its relatively small size (27 kDa) as well as the poorly intracellular delivery efficiency.10,14 To address these problems, a recombinant cellpenetrating TCS (termed rTL) was designed, in which lowmolecular-weight protamine (LMWP) was fused to TCS. LMWP is a potent cationic cell-penetrating peptide (CPP) that has been well demonstrated in our previous works.15−18 In

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RESULTS

Protein Expression. TCS is a type I ribosome-inactivating protein that lacks active mechanisms for cell entry thus exhibiting poor cell permeability. The plasmid pTXB1-TCSLWMP was constructed to express the rTL fusion protein.22 The purified rTCS or rTL showed an approximate molecular weight of 27 or 29 kDa, respectively (Figure S1A, Supporting Information). The rTL was then characterized using matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), showing the molecular weight to be about 29 kDa (Figure S1B). The purity of rTL was further assessed by a desalting column and heparin-affinity column, with a purity of over 90% (Figure S1C,D). 26649


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Figure 1. (A) Size and TEM of the rTL-bound NPs. (B) Size and TEM of the rTL/ABZ@BSA/Ag NP. (C) Zeta potential of the rTL/BSA NPs with different ratios. (D) The rTL/ABZ@BSA NPs at a ratio of 5:1 and (E) the rTL/ABZ@BSA/Ag NPs at a ratio of 5:1. (F) In vitro release of ABZ.

Figure 2. Cellular uptake of the rTL/ABZ@BSA/Ag NPs in A549/T and HCT8/ADR cells. (A) Fluorescence images of the cells treated with the NPs labeled with coumarin-6. (B) FACS analysis of the uptake efficiency of rTL/ABZ@BSA NPs. (C) FACS analysis of the uptake efficiency of rTL/ABZ@BSA/Ag NPs. (D) Quantitative analysis of fluorescence intensity. (E) Cellular uptake of the rTL/ABZ@BSA/Ag NPs in HCT8/ADR cells. Fluorescence images of the cells treated with the NPs with coumarin-6. (F) FACS analysis of the uptake efficiency of rTL/ABZ@BSA NPs. (G) FACS analysis of the uptake efficiency of rTL/ABZ@BSA/Ag NPs. (H) Quantitative analysis of fluorescence intensity. Scale bar, 50 μm.

Characterization of rTL-Bound NPs. TCS is a cationic protein, with an isoelectric point of 9.4, containing no carbohydrate, phosphorus, and cysteine. Table S1 presents the particle size, polydispersity index, zeta potential, drugloading efficiency, and drug-loading capacity of the rTL/ABZ@ BSA NPs and rTL/ABZ@BSA/Ag NPs. Both NPs showed narrow size distribution and negative ζ-potential. The ABZ@

BSA NPs and ABZ@BSA/Ag NPs were also characterized (Figure S1E). The co-delivery system was characterized by the formation of self-assembling structures via stable electrostatic interaction between the cationic rTL and the anionic bovine serum albumin (BSA) NPs. The rTL binding capacity was shown in Figure S1F. The NPs remained stable at room temperature for 24 h (Figure S1G). The size of rTL/ABZ@ 26650


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Figure 3. Cellular uptake of the Rhodamine B-labeled rTL and rTL/ABZ@BSA/Ag NPs in A549/T and HCT8/ADR cells. (A) Fluorescence images of the cells treated with the NPs. (B) FACS analysis of the uptake efficiency of the NPs. (C) Quantitative analysis of fluorescence intensity. (D) Western blotting analysis of the SPARC expression in A549/T cells and tumor. (E) Cellular uptake of the Rhodamine B-labeled rTL and rTL/ ABZ@BSA/Ag NPs in HCT8/ADR cells. Fluorescence images of the cells treated with the NPs. (F) FACS analysis of the uptake efficiency of the NPs. (G) Quantitative analysis of fluorescence intensity. (H) Western blotting analysis of the SPARC expression in HCT8/ADR cells and tumor. Scale bar, 50 μm.

Figure 4. Antitumor activity (IC50) of the rTL NPs in drug-resistant cell lines of (A) A549/T and (B) HCT8/ADR.

In Vitro Cellular Uptake. The cellular uptake of the rTLbound NPs was investigated in the MDR cancer cell lines by using the dye-labeled BSA NPs. Both the rTL/ABZ@BSA NPs and rTL/ABZ@BSA/Ag NPs displayed a higher cellular uptake efficiency than that of the ABZ@NPs, according to the fluorescence microscopic observation and flow cytometry assay in the A549/T (Figure 2A−D) and HCT8/ADR cells (Figure 2E−H). Of interest, the binding with the albumin-

BSA NP was about 295 nm, whereas that of rTL/ABZ@BSA/ Ag NP was about 105 nm; both were negatively charged (Figure 1A,B;D,E). With the increasing amount of rTL, the negative zeta potential of the rTL/BSA NPs was reduced (Figure 1C). An optimized mass ratio of rTL/ABZ@BSA NPs (5:1) was used for further studies. The ABZ release profile is shown in Figure 1F. 26651


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Figure 5. Apoptosis assay using flow cytometry. Flow cytometry scatterplots (A) and quantitative analysis of the total apoptosis rate (B) in A549/T cells. Flow cytometry scatterplots (C) and quantitative analysis of the total apoptosis rate (D) in HCT8/ADR cells.

The p38-MAPK inhibitor SB203580 is able to suppress the expression of SPARC.27 In the presence of SB203580, the results revealed that the cellular uptake of the rTL-bound NPs was reduced, in accordance with the downregulation of the SPARC expression (Figure S2A,B). In Vitro Cytotoxicity and Cell Apoptosis Assays. The A549/T and HCT8/ADR cells were highly resistant to the chemo drugs such as paclitaxel and doxorubicin, and our previous investigation showed that the cell viability was not affected even at a high PTX concentration of up to 20 μg/mL in the A549/T cells.13 By contrast, the rTL-bound NPs displayed a high antitumor activity in a dose-dependent manner, with the IC50 of 0.14 and 0.08 μg/mL (ABZ) in the A549/T cells for the rTL/ABZ@BSA NP and rTL/ABZ@ BSA/Ag NP, respectively (Figure 4A), whereas it was 0.13 and 0.09 μg/mL (ABZ) in the HCT8/ADR cells, respectively (Figure 4B, Table S2). The rTL-bound NPs exhibited superior efficacy than the single use of rTL or the ABZ-encapsulated NPs. The antitumor activity of the NPs on the nonresistant tumor cells (A549, HCT8) was shown in Figure S3A. The NPs displayed less cytotoxicity in the nontumoral cells (HUVEC), suggesting the biocompatibility (Figure S3B). Furthermore, an Annexin V-FITC and propidium iodide (PI) method was used to measure the cells in early apoptosis (FITC+/PI−), late apoptosis (FITC+/PI+), and necrotic cells

based NPs did not compromise the uptake efficiency of the cellpenetrating rTL; instead, a slight increase for the rTL-bound NPs was observed. The cellular uptake efficiency of rTL labeled with rhodamine B isothiocyanate was also investigated. The uptake profile of rTL was similar to that of the NPs (Figure 3). The results suggested synergistic intercellular delivery via the CPP- and albumin-binding protein-mediated pathways. Albumin-binding proteins were overexpressed on tumor cells and tumor vascular endothelial cells.23,24 In our previous study, the albuminbinding protein SPARC was found with overexpression on glioma, colon cancer, and tumor vessel endothelium, which promoted the enhanced cellular uptake of albumin nanoparticles.16,25 The high expression of SPARC in the A549/T cells and the xenografted tumors was demonstrated using Western blotting (Figure 3D), and the same results were found in the HCT8/ADR cells and the tumor (Figure 3H). It is noteworthy that it has been reported that induction of clusters of albumin-binding proteins on cell membranes is an important step for triggering albumin transportation.26 The albumin NPs could be potent inducers for clustering the albumin-binding proteins because the NPs could bind with cross-linked multiple receptors thus displaying an efficient way to trigger the transportation. 26652


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Figure 6. Bivariate JC-1 analysis of the MMP in A549/T cells (A) and HCT8/ADR cells (B) by flow cytometry. Western blotting analysis of the caspase 3 level (C), procaspase 9 and caspase 9 phosphorylation levels (D), caspase 9 activity measurement (E).

Figure 7. Change of morphology of the A549/T cells. Scale bar, 50 μm.

(FITC−/PI+). As shown in Figure 5A,B, the single use of free PTX, ABZ, or rTL induced a low apoptosis rate in the A549/T cells. A significant increase of the total apoptosis rate was observed in the cells treated with the rTL/ABZ@BSA NPs or rTL/ABZ@BSA/Ag NPs, showing 41.6 or 64.5%, respectively. By contrast, the apoptosis rate of the ABZ-encapsulated NPs (without rTL) was about 28%. Similar results were seen in the HCT8/ADR cells (Figure 5C,D), in which the apoptosis rates were 20.7 and 48.5% for the rTL/ABZ@BSA NPs and rTL/ ABZ@BSA/Ag NPs, respectively, which is significantly higher than that of other groups. The results confirmed that the protein toxin TCS and the small drug ABZ could yield a

synergistic effect in the MDR cells via a mechanism of enhanced apoptosis. The mitochondrial pathways and caspase family play a primary role in chemotherapy-induced apoptosis.28 In our studies, the mitochondrial membrane potential (MMP) was monitored. A shift from the highly energized mitochondrion (high ΔΨm) to low-voltage mitochondrion (low ΔΨm) indicates the collapse of the mitochondrial membrane and initiation of apoptosis. After treatment, the low ΔΨm ratios in the A549/T cells were 36 and 47% for the rTL/ABZ@BSA NPs and rTL/ABZ@BSA/Ag NPs, respectively (Figure 6A), whereas those in the HCT8/ADR cells were 81 and 88%, respectively (Figure 6B). The minor enhancement of the rTL/ 26653


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Figure 8. Effects on the skeleton in A549/T cells. (A) FACS analysis of α-tubulin when treated with the rTL NPs or free drugs. (B) Quantitative analysis of fluorescence intensity. (C) Cell-cycle analysis. (D) Quantitative results of cell-cycle phases analyzed by flow cytometry, each experimental group was compared with the phosphate-buffered saline (PBS) group. (E) Western blotting analysis of the level of β-tubulin (s)-dissociation and βtubulin (p)-polymerization. (*P < 0.05, **P < 0.01, in comparison with those of the PBS group).

ylation of caspase 9.13 We found that procaspase 9 levels were decreased by the rTL/ABZ@BSA/Ag NPs and caspase 9 (pT125) phosphorylation was inhibited and the phosphorylated caspase 9 (pS144) was also down-regulated (Figure 6D). Moreover, caspase 9 activity was significantly increased after the treatment with the rTL/ABZ@BSA NPs (Figure 6E). Cytoskeleton and Cell Cycle Affected by rTL-Bound NPs. The cytoskeleton is a dynamic structure essential for a wide variety of normal cellular processes, including the maintenance of cell morphology and movement.30 Apoptosis is a programmed process with the unique change of morphology. For example, paclitaxel can arrest the tumor cells in mitosis and induce apoptosis, and the cells become multinucleated.31 Usually, the appearance of nuclear fragmen-

ABZ@BSA/Ag NPs could be accounted for by the Ag NPssynergized effect because Ag NPs can also impair mitochondrial functions and induce apoptosis.29 The enhanced apoptosis mediated by the rTL-bound NPs was further confirmed by Western blotting (Figure 6C). The rTL-bound NPs efficiently activated procaspase 3 and increased the transformation of procaspase 3 to the activated caspase 3. The rTL/ABZ@BSA/Ag NPs exhibited the highest activation efficiency. Caspase 9 plays a crucial role in apoptosis; its activation can be inhibited by phosphorylation at multiple sites. Our previous study showed that drug resistance in A549/T cells was associated with upregulation of caspase 9 phosphorylation and TCS could resensitize the MDR cells through dephosphor26654


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Figure 9. (A) Cell-cycle analysis on HCT8/ADR cells. (B) Quantitative results of cell-cycle phases analyzed by flow cytometry. Each experimental group was compared with PBS group. (C) Western blotting analysis of the level of P-gp. (*P < 0.05, **P < 0.01, in comparison with the PBS group).

pointed out that the reduction of the P-gp level was moderate and further investigations should be conducted to elucidate the detailed mechanisms. The inhibition of tubulin was further investigated with αtubulin antibody (red), βIII-tubulin antibody (green), and 4′-6diamidino-2-phenylindole (DAPI; blue) staining. The immunofluorescence observation showed that the cells treated with the rTL-bound NPs become multinucleated and disorganized the microtubule network, compared with the untreated cells that displayed long filamentous microtubule distribution across the cells (Figure 10). The rTL-bound NPs caused a formation of bundles of short microtubules around the edges of the cells. In Vitro Wound-Healing Assay and Transwell Migration Assays. The migration and invasion of cancer cells are closely related to the cytoskeleton.3 A wound-healing assay was used to investigate the A549/T cell migration and cell interactions. As shown in Figure 11A, the A549/T cells displayed the high metastatic ability and the wound closure was observed at 24 h. However, by treatment with the rTL/ABZ@ BSA/Ag NPs, the gap remained open, indicating the inhibition of cell migration. The anti-invasive effect of the rTL/ABZ@ BSA/Ag NPs on A549/T cells was examined by Matrigel invasion assays. The rTL/ABZ@BSA/Ag NPs exerted a strong inhibitory effect on A549/T cell migration, consistent with the wound-healing results (Figure 11B). RIPs are the potential drugs for antimetastasis.36 TCS can inhibit tumor growth by inducing apoptosis of tumor cells and suppress cell migration and metastasis.11,37 The effect on the cytoskeleton could be a possible mechanism. In Vivo Treatment Efficacy. The antitumor effect was further evaluated in the nude mice bearing A549/T tumors. The results showed that both the rTL-bound NPs yielded high antitumor activity and effectively arrested the growth of the drug-resistant tumor, with tumor growth inhibition rates of 57% (rTL/ABZ@BSA NPs) and 86% (rTL/ABZ@BSA/Ag NPs) (Figure 12A−C). The significant enhancement of antiMDR tumor efficacy in the rTL/ABZ@BSA/Ag NPs group was due to the cocktail treatment containing three active compounds in the rTL/ABZ@BSA/Ag NPs via synergistic

tation in the cells is used as an indicator of late-stage apoptosis and the important cellular event, the point-of-no-return in apoptosis.32 As shown in Figure 7 (red arrow), the treatment with the rTL/ABZ@BSA/Ag NPs caused nuclear fragmentations, such as karyopyknotic, karyorrhectic, multinucleate, and vacuoles, indicating the point-of-no-return in apoptosis. The antitumor effect of ABZ is related to its inhibition of tubulin polymerization.33 TCS also possesses antitubulin polymerization activity.12,34 Microtubules are formed by the polymerization of α-tubulin and β-tubulin and are the important components of cytoskeleton for maintaining the structure of the cell.35 Our results showed that the level of αtubulin in the rTL/ABZ@BSA/Ag NP-treated group decreased significantly compared to that of other groups (Figure 8A,B), as measured by using the Tubulin-Tracker Red assay. In addition, the treatment with the rTL/ABZ@BSA/Ag NP also inhibited the polymerization of β-tubulin (Figure 8C). The antitumor effect of ABZ is related to its effect on G2/M phase arrest.33 TCS can induce apoptosis via a mechanism of G1 phase arrest.11 Our studies also demonstrated that rTL caused G1 phase arrest in the A549/T cells (Figure 8D,E). An elevating ratio at the G2/M stage was observed when the cells were exposed to ABZ-containing NPs. The percentage of the G2/M phase in the group of rTL/ABZ@BSA/Ag NPs was much higher than that in the group of the ABZ@BSA/Ag NPs (42.64 vs 26.59%), suggesting the synergistic effect of rTL and ABZ. In the HCT8/ADR cell lines, there were similar trends of the enhanced cell-death induction activities of the rTL/ABZ@ BSA/Ag NPs (Figure 9A,B). These results further confirmed that the rTL/ABZ@BSA/Ag NPs had an obvious inhibitory effect on spindle formation and mitosis (cell-cycle arrest). Therefore, it was reasonable to deduce that the rTL/ABZ@ BSA/Ag NPs involved multiple mechanistic interplays among the destruction of the cytoskeleton, apoptotic induction, and arrest of the cell cycle, thus yielding a synergistic therapeutic effect. Interestingly, it also showed a downregulation of the Pgp expression in the HCT8/ADR cells treated with the rTL/ ABZ combination (Figure 9C). The inhibition of P-gp was beneficial for reversing MDR. However, it should also be 26655


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Figure 10. Confocal images of cells labeled α-tubulin antibody (red), βIII-tubulin antibody (green), and DAPI (blue). A significant increase in apoptotic nuclear fragmentation occurred in the cells treated with rTL/BSA NPs, compared to that in PBS, rTL, and BSA NPs.

actions. The antitumor and anti-MDR activity of Ag NPs was also demonstrated in our previous studies.8,29 The EPR effect-

associated tumor distribution could play a role in enhanced treatment efficacy. By contrast, the monotherapy using the 26656


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Figure 11. Cell migration and invasion studies using A549/T cells. (A) Wound-healing assays. (B) Transwell migration assays with staining by crystal violet. Scale bar, 100 μm.

S4B). Moreover, the mice treated with rTL exhibited obvious weight loss, which could be accounted for by its nonspecific cell penetration thus causing the unwanted toxicities. By contrast, no significant weight loss was found in other groups, demonstrating the reduced side toxicities (Figure 12D). The histopathological examination revealed the liver- and kidneyassociated toxicity in the mice treated with rTL, whereas no

ABZ@BSA NPs did not carry sufficient treatment efficacy at the tested dose. Although rTL possessed high antitumor activity in vitro, its rapid clearance compromised the in vivo treatment outcomes.22 Compared with the NPs, the faster clearance of rTL was also observed in our studies (Figure S4A). But both the rTL and the NPs showed effective accumulation inside the tumor (Figure 26657


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Figure 12. Antitumor efficacy of the subcutaneous A549/T tumor-bearing mice model. (A) Tumor growth curve. (B) Tumor weight. (C) Tumor tissues. (D) Changes in body weight during the course of treatment.

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DISCUSSION MDR and metastasis are two pivotal challenges for cancer treatment.38 Here, we reported a novel nanotherapy with codelivery of a protein and small drug (TCS and ABZ) via albumin-coated silver NPs for overcoming MDR and metastasis via multiple mechanisms. The rTL/ABZ@BSA/Ag NPs were demonstrated to induce apoptosis efficiently. Our results revealed that the mitochondrial apoptotic pathway was an important mechanism responsible for the combination therapy of TCS and ABZ. The Ag NPs could also efficiently inhibit tumor-cell growth via the mitochondrial apoptotic pathway.24 Hence, due to the synergistic effect of Ag NPs, the rTL/ABZ@ BSA/Ag NPs exhibited a greater shift toward low ΔΨm and induced a significantly higher apoptosis rate compared to that of the rTL/ABZ@BSA NPs. The apoptotic event of nuclear fragmentation is an important cellular event indicating the destruction of the cytoskeleton30 and the point-of-no-return in cell apoptosis.32 In our study, we confirmed that the combination therapy using the rTL/ABZ@ BSA/Ag NPs could reach the point-of-no-return in apoptosis, with enhanced activation of caspase 3, thus causing irreversible cell apoptosis and efficient cell death. The phosphorylation of apoptosis-associated caspase 9 is related to drug resistance. The treatment with the rTL/ABZ@BSA/Ag NPs led to the suppression of caspase 9 phosphorylation (pT125 and pS144). The cytoskeleton is essential for the maintenance of the cell shape and morphology. Microtubules are an important component of the cytoskeleton, which form the mitotic spindle

obvious pathological change was found in the groups receiving the rTL/ABZ@BSA NPs and rTL/ABZ@BSA/Ag NPs (Figure 13). However, there were minor changes in the lungs from the mice given the rTL and the combination groups, and the potential side effects in lungs should be further investigated. At the endpoint, lung tissues from each group were collected and the number of metastatic nodules from each lung was recorded to evaluate the antimetastasis efficacy. The subcutaneous A549/T tumor exhibited a high incidence of metastasis to the lung, and many metastatic nodules were observed in the lungs from the groups of PBS and ABZ. However, the number of metastatic nodules in the groups treated with the rTL/ ABZ@BSA NPs and rTL/ABZ@BSA/Ag NPs dramatically decreased by 85.2 and 95.1%, respectively, compared with the PBS control group (Figure 14). The tumor cells were observed in the groups of saline, ABZ@BSA NPs, and ABZ@BSA/Ag NPs, respectively, in the histopathological examination (Figure 13). The results were consistent with the wound-healing assay and Transwell migration assay. The TUNEL results showed that the rTL/ABZ@BSA/Ag NPs effectively induced apoptosis in the tumor, and the combination therapy displayed superiority over the monotherapy of rTL and ABZ NPs (Figure 15A). The Western blotting analysis of caspase 3 in A549/T subcutaneous tumor further confirmed that the rTL/ABZ@BSA/Ag NPs showed the highest capacity among the tested groups (Figure 15B). The results of TUNEL and caspase 3 assays were consistent. 26658


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Figure 13. Histopathological examination of major organs collected at the experimental endpoint. Tumor cells were found in the saline, ABZ@BSA NP and ABZ@BSA/Ag NP groups. Minor changes in lungs were found in the rTL and combination groups. The rTL group showed the liver and kidney-associated toxicity, including disarrangement, focal necrosis and vacuolation of hepatic cells in the liver, tubules dilated, protein tube type, and glomerular fibrosis in kidney. The rTL/ABZ@BSA NPs and rTL/ABZ@BSA/Ag NPs showed no pathological change. Scale bar, 50 μm.

Figure 14. (A) Quantitative analysis of pulmonary metastatic nodules for every group in A549/T cells. (B) The collected lungs for every group in A549/T cells.

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Figure 15. Detection of apoptosis in tumors. (A) TUNEL assay. (B) Western blotting.

and mediate chromosome segregation during cell division. Microtubule is the target for many anticancer drugs (e.g., paclitaxel and vincristine) via a mechanism of inhibition of the dynamic assemblies of tubulin. It has been reported that both TCS and ABZ can possess antitubulin polymerization activity.12,33,34 These drugs can block mitosis, inhibit cell proliferation, and induce cell death through apoptosis. The synergistic actions of TCS and ABZ yielded a significant impact on the cytoskeleton. The level of α-tubulin was reduced by treatment with the rTL/ABZ@BSA/Ag NPs. It was a possible anti-MDR mechanism because a high level of α-tubulin could down-regulate the sensitivity of tumor cells.39 Furthermore, the combination of rTL/ABZ@BSA/Ag NPs also inhibited the polymerization of β-tubulin and caused a structural change of the microtubule network. The suppression of the P-gp function can enhance the efficacy and cytotoxicity of a chemotherapeutic. The treatment of the rTL/ABZ@BSA/Ag NPs resulted in the downregulation of P-gp. In addition, it should be noted that high expressions of drug pumps can promote tumor metastasis.40,41 Pulmonary metastasis often occurrs in many solid tumors; the metastasis through lymph nodes is a usual mechanism. A549/T is a lung cancer cell line. Therefore, the metastasis from the xenograft implantation could be preferentially homing to the lung but not to other organs. The phenomenon is interesting, and we suspected that the metastasis was associated with the enhanced cell migration and invasion ability of the A549/T cells, possibly due to the cytoskeleton changes. In the studies, we found that the subcutaneous A549/T tumor model exhibited a high incidence of lung metastasis. An interesting find of our work was that the combination of TCS and ABZ inhibited the metastasis efficiently. The TCS-contained treatment could arrest the metastasis with an inhibition efficiency >80% compared to that of the control group. More

importantly, the TCS-containing groups were significantly higher than the ABZ-alone groups, suggesting that TCS could be the major mechanism for antimetastasis. The actions on the cytoskeleton were not only responsible for reversing drug resistance but also associated with antimetastasis. Microtubules play an important role in tumor-cell migration, invasion, and metastasis.3 It has been revealed that there is a tight correlation between a high level of acetylated α-tubulin and aggressive metastasis.42 The antimetastasis mechanism of TCS could be also accounted for the cytoskeleton-associated effect. However, more detailed investigations on this issue still need to be conducted to help better understand the mechanisms.

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CONCLUSIONS In summary, we designed a novel nanotherapy for co-delivery of a protein drug TCS and a small drug ABZ via the albumincoated silver NPs. The rTL/ABZ@BSA NPs exhibited an efficient antitumor effect and arrested metastasis via multiple mechanisms, such as enhanced apoptosis, downregulation of Pgp, and dysfunction of the cytoskeleton. In the drug-resistant A549/T lung tumor model, the rTL/ABZ@BSA NPs resulted in a significant inhibition of tumor growth and efficient prevention of lung metastasis. Therefore, this work illustrated a novel strategy in the development of nanomedicine-based combination therapy for overcoming both drug resistance and tumor metastasis.

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EXPERIMENTAL SECTION

Materials. Recombinant trichosanthin plasmid pET28a-TCS was a kind gift from Prof. Pang-Chui Shaw (Chinese University of Hong Kong). The pTXB1 vector, including the IMPACT (intein-mediated purification with affinity chitin-binding tag) system, were obtained from New England Biolabs (England). The lysogeny Broth (LB) medium was purchased from Oxoid (England). Protein marker and 26660


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ACS Applied Materials & Interfaces isopropyl b-D-1-thiogalactopyranoside (IPTG) were obtained from Thermo. Bradford, BCA microplate protein assay kit and tubulintracker red assay kit were obtained from Beyotime Institute of Biotechnology (Haimen, China). Fetal bovine serums (FBSs) and 1640 cell culture medium were purchased from Gibco. The antibiotics were acquired from Amresco. L-Cysteine was obtained from J&K Scientific Ltd (Shanghai, China). The JC-1 dye was from Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). Albendazole and NHS-Cy5 were obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Anticaspase 3 monoclonal antibody (Cat: 9662) and Cleaved Caspase 3 monoclonal antibody (Cat: 9664) were purchased from Cell Signaling Technology. Antimouse P-gp was purchased from Abcam and anti-mouse β-actin was purchased from Sigma-Aldrich. All other reagents were of analytical grade from Sinapharm Chemical Reagent Co., Ltd. (Shanghai, China). Construction of the Recombinant TCS-LMWP (Termed rTL). The rTL expression vector pTXB1-TL (pTL) was constructed by PCR using pET-TCS vector (pET28a-TCS) as the template. The coding sequence of LMWP was fused to the 3′ end of the TCS fusion gene and then cloned to the prokaryotic expression vector pTXB1, at the site NdeI/XhoI. Expression and Purification of Protein rTL. The production method of rTL was modified from our previous method.22 In brief, a single colony of BL21(DE3) E. coli transformed with pET-rTL was inoculated in an LB medium containing 100 μg/mL ampicillin at 37 °C, with shaking at 250 rpm. The expression of rTL was then induced by addition of IPTG (0.3 mM) and overnight incubation at 25 °C, with shaking at 150 rpm. The E. coli cells were harvested by centrifugation and re-dispersed in the column buffer (20 mM N-(2hydroxyethyl)piperazine-N′-ethanesulfonic acid, 500 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.05% Tween 20, pH 8.5) and lysed by sonication. The supernatant fraction containing the soluble rTL was collected and loaded onto the Chitin resins. After a thorough wash, induction of the on-column cleavage was conducted by quickly flushing the column with 3-bed volumes of the cleavage buffer (the column buffer containing 50 mM cysteine), followed by incubation at 4 °C for 16 h for on-column cleavage to yield the protein rTL. rTLs were characterized by using FPLC (Ä KTApurifier 10, GE Healthcare) equipped with the heparin-affinity column based on the interaction of LMWP and heparin (GE Healthcare). The expression of rTL was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and MALDI-TOF-MS (MALDI TOF/TOF 5800 analyzer, AB Sciex, Framingham, MA). The final concentration of the rTL product was determined by the BCA assay. Preparation of Albendazole-Loaded Albumin Nanoparticles. The preparation method was described as follows. In brief, 400 μL of TCEP·HCl solution (32 mg/mL) was added to 16 mL of BSA solution (2.5 mg/mL, in 12.5 mM Tris buffer) under stirring. Afterward, 4 mg of ABZ in 800 μL of tetrahydrofuran was mixed with the BSA solution under constant magnetic stirring at 1200 rpm for 2 h. The mixture was then sonicated at 4 °C, followed by dialysis for 1 h against water. The thus-formed nanoparticles were collected by centrifugation at 8000 rpm for 20 min at 4 °C and filtered through a 0.22 μm membrane. The ABZ concentration was measured by ultraperformance liquid chromatographic (UPLC) system (ACQUITY UPLC, Waters). The UPLC analysis of ABZ was performed using a C18 BEH column (1.7 μm, 2.1 mm × 50 mm, Phenomenex, CA), with a mobile phase consisting of acetonitrile and ultrapure water (20:80, v/ v), at a flow rate of 0.3 mL/min. The effluents were monitored at 294 nm, and the concentration was calculated with a calibration curve. Preparation of ABZ@BSA/Ag NPs and rTL/ABZ@BSA/Ag NPs. Nanosilver (Ag NP) was synthesized following the procedures described.43 In brief, 16 mL of glucose solution (2.5 mg/mL) was mixed with 800 μL of silver seed solution and stirred for 5 min at 75 °C. To this solution, 16 mg of AgNO3 in 1.6 mL of water was added dropwise under vigorous stirring. The ABZ-loaded albumin-silver nanoparticles were prepared as above, with a minor modification in procedures by adding ABZ first and 10 mL of Ag NPs subsequently.

The encapsulation efficiency and drug-loading capacity of the nanoparticles were measured by UPLC and calculated using the following formula

encapsulation efficiency (%) weight of encapsulated drug = × 100% weight of drug added drug‐loading capacity (%) weight of encapsulated drug = × 100% weight of NPs Stability of Nanoparticles and Drug Release in Vitro. The BSA NPs were dispersed in PBS (pH 7.4) to investigate the nanoparticle stability. The turbidity assay was used for indicating colloidal stability. The in vitro release tests from NPs in the release medium (PBS, pH 7.4, containing 0.5% w/v tween 80) were conducted using the dialysis method (molecular weight cutoff 10−12 kDa) in a 37 °C shaker. At the predetermined time points, 0.5 mL of the dissolution medium was taken for measurement and replenished with an equivalent amount of fresh release medium. The released amount of drugs was determined by UPLC. Through an electrostatic interaction between the cationic rTL and anionic ABZ@BSA NPs (or ABZ@BSA/Ag NPs), the nano-selfassembly (rTL/ABZ@BSA NPs or rTL/ABZ@BSA/Ag NPs) was formed by incubation for 1 h at 37 °C. rTL was complexed with the ABZ@BSA NPs at various ratios, and the optimized ratio was a 5-fold molar excess of rTL. The particle size and zeta potential were measured using ZetaSizer Nano-ZS90(Malvern, U.K.). Cellular Studies. Cell Lines. The human colon cancer cell line HCT8 and human nonsmall cell lung carcinoma A549 were obtained from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). The drug-resistant HCT8/ADR and A549/T were a kind gift from Prof. Ding Jian at SIMM, CAS. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (100 μg/mL streptomycin and 100 μg/mL penicillin) at 37 °C in 90% relative humidity and 5% CO2. In Vitro Uptake Studies. The cells were seeded in a 12-well plate at a density of 1 × 105 cells/well and cultured for 24 h. The nanoparticles labeled with coumarin-6 were added to the cells for a 2 h incubation. The cells were then washed with PBS three times, fixed with 4% paraformaldehyde, and stained with DAPI. Fluorescent images were observed using a fluorescence microscope (Zeiss, Germany). In addition, the cellular uptake efficiency was determined using a flow cytometer (BD Pharmingen). In addition, rTL was labeled with rhodamine B isothiocyanate to investigate its uptake efficiency. In Vitro Cytotoxicity Studies. The cell viability was measured by a standard (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. A549/T, HCT8/ADR, and HUVEC were seeded in a 96-well plate at a density of 5000 cells per well. After 24 h, the nanoparticles were given to the cells, followed by incubation for 48 h. The concentration of NPs was up to 3.2 μg/mL (equal to 3.2 μg/ mL of ABZ). The cell viability was measured using a standard MTT method and calculated using a formula as follows.

cell viability (%) = (ODtest − ODDMSO)/(ODcontrol − ODDMSO) × 100% Furthermore, the cell apoptosis was quantitatively measured using FACS (BD Pharmingen). The cells were seeded in a 6-well plate and allowed to grow for 24 h. The cells were exposed to the drugs (in A549/T and A549, a dose equal to 0.5 μg/mL of ABZ and 2.5 μg/mL of rTL; in HCT8/ADR and HCT8, a dose equal to 2 μg/mL of ABZ and 10 μg/mL of rTL; PTX 0.5 μg/mL for A549/T and 2 μg/mL for HCT8/ADR), with a subsequent 48 h incubation. The cells were collected and washed with PBS three times and stained with an FITC Annexin V apoptosis detection kit (BD Pharmingen) according to the manufacturer’s protocol. The cells were subjected to apoptosis analysis using FACS. In addition, the cells treated with the drugs for 24 h were 26661


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ACS Applied Materials & Interfaces also collected for caspase 9 and caspase 3 detection using Western blotting. Caspase 9 Activity Detection. The caspase 9 activity of untreated A549/T cells, cells treated with rTL, BSA NPs, and rTL/BSA NPs with 2.5 μg/mL rTL for 8 h were measured using a caspase 9 activity assay kit (Beyotime Institute of Biotechnology, China), following the manufacturer’s instructions. Tubulin Assay. Tubulin-Tracker Red (α-tubulin Tracker Red) was used to measure the α-tubulin level in the A549/T and HCT8/ADR cells. The cells treated with the drugs were harvested for the α-tubulin Tracker (red) assay according to the manufacturing’s protocol and analyzed by flow cytometry. In addition, the cells treated with the drugs were also collected for β-tubulin detection using Western blotting, as previously described. Tubulin polymerization assay: Briefly, the cells were lysed with RIPA containing protease inhibitors. The samples were centrifuged at 13 000g for 10 min at 37 °C, and the supernatant containing depolymerized tubulins was transferred to a new tube. The pellet (polymerized tubulin) was resuspended in an equal amount of hypotonic buffer before the Western blot assay. Immunofluorescence: The cells grew on sterile coverslips embedded in a 24-well plate. A549/T cells were incubated with or without rTL/ BSA NPs for 4 h and fixed with ice-cold methanol. Samples were blocked with 5% BSA in PBS. The cell slices were incubated overnight at 4 °C with anti-α-tubulin and anti-βIII-tubulin antibody and subsequently with Alexa Fluor 647-conjugated goat anti-mouse IgG antibody and Alexa Fluor 488-conjugated goat antirabbit IgG secondary antibody for 1 h at room temperature, followed by DAPI staining. The co-localization of NPs with α-tubulin and anti-βIIItubulin was observed by laser scanning confocal microscopy (TCS− SP8; Leica, Germany). Morphology of Cancer Cells. The A549/T cells were seeded in a 12-well plate at a density of 1 × 105 cells/well and cultured for 24 h. The cells were treated with the drugs, followed by incubation for 48 h. The cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. The cell nuclei were stained with DAPI for 10 min. Fluorescent images were observed with a fluorescence microscope. Cell Cycle. For cell cycle analysis, the cells were fixed with 70% ethanol at 4 °C overnight and thus stained with the PI solution (50 μg/mL PI, 50 μg/mL RNase A, 0.1% Triton X-100, and 37 μg /mL EDTA) at 4 °C for 30 min. FlowJo_V10 software (Treestar, Inc., San Carlos, CA) was used to analyze the proportion of every phase. Mitochondrial Membrane Potential Assay. A shift from the highly energized mitochondrion (high ΔΨm) to low-voltage mitochondrion (low ΔΨm) can be measured by the JC-1 dye, with fluorescence change from red to green, thus providing a quantitative index of apoptosis for the flow cytometry assay.27 The cells were treated with the drugs for 3 h and then incubated with 5 μM of JC-1 for 30 min. The cells were washed twice with prewarmed PBS, harvested, and analyzed using a flow cytometer. Wound-Healing Assay. The A549/T cells at 90% confluency were scratched with a pipette tip, and the floating cells were washed away with PBS. The cells were then treated with the drugs at a dose equal to 0.25 μg/mL of ABZ, equal to 1.25 μg/mL rTL). The gap closure was observed at 8 and 24 h under a microscope. Cell Migration and Invasion Assays. Cell migration and invasion assays were performed using Transwell chambers (6.5 mm, 8 μm pore size, Corning), coated with the Matrigel matrix (Corning). In brief, the cells were seeded in the upper chamber of a Matrigel-coated chamber. After 12 h culture, the cells were treated with the drugs at a dose equal to 0.25 μg/mL ABZ and 1.25 μg/mL rTL. After 48 h, the cells that migrated to the lower surface were fixed, stained with crystal violet for 30 min, and counted under a microscope. In Vivo Therapeutic Efficacy on Tumor Growth and Lung Metastasis. In Vivo Antitumor Therapy. The animal study procedures were approved by the Institutional Animal Care and Use Committee, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences. (Note: SIMM is an institution with AAALAC International accreditation). The studies were conducted on female

Balb/c-nu nude mice aged 3−4 weeks. The drug-resistant A549/T cells were subcutaneously implanted (1 × 106 cells/mouse) on the back. The mice were divided into six groups randomly (six per group): PBS, ABZ@BSA NPs, ABZ@BSA/Ag NPs, rTL, rTL/ABZ@BSA NPs, and rTL/ABZ@BSA/Ag NPs. When the tumor volume reached approximately 100 mm3, the animals were treated with the NPs at a dose equal to 1.5 mg/kg of ABZ and 2.5 mg/kg of rTL via tail vein injection once per 2 days in a period of 20 days. The tumor volume and body weight were monitored. The tumor volume was calculated using the following formula

V = (L × W 2)/2 At the experimental endpoint, the mice were sacrificed and the tumors and major organs were harvested for histological examination. The metastatic nodules on the pulmonary tissues were counted, and the lung sections were stained with hematoxylin and eosin (H&E) to further evaluate the metastasis. The tumor tissues were used for TUNEL detection. In Vivo Imaging. The mice bearing subcutaneous A549/T were given the rTL-Cy5 and rTL-Cy5/BSA NPs via tail vein injection. The biodistribution was monitored using an IVIS imaging system (Caliper PerkinElmer; Hopkinton, MA) at the predetermined time points. At the endpoint of the experiment, the tumor tissues were harvested for the preparation of cryosection to 20 μm slices (CM1950, Leica, Germany). The tissue slices were stained with DAPI. The distribution of the Cy5-labeled rTL was observed by confocal laser scanning microscopy. Statistical Analysis. Statistical analysis was performed by t-test and one-way ANOVA. Each experiment was performed in triplicate, and data are represented as mean ± standard deviation. Statistically significant differences were defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b05292. Additional figures and table (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Q.X.). *E-mail: [email protected] (J.W.). *E-mail: [email protected] (Y.H.). ORCID

Yongzhuo Huang: 0000-0001-7067-8915 Author Contributions ⊥

Y.T. and J.L. have contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Basic Research Program of China (973 Program 2014CB931900, 2013CB932503) and NSFC, China (81373357, 81422048, 81673382, and 81521005) for the financial support.

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REFERENCES

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Co-Delivery of Trichosanthin and Albendazole by Nano-Self-Assembly

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